Marijuana is currently the most widely abused street drug. However, the functional significance of the cannabinoid receptor system in health and disease includes the use of cannabinoids as analgesics, antiemetics in cancer patients, anticonvulsant for epilepsy and as antiglaucoma agents as well as immunomodulatory agents. These diverse pharmacological effects of cannabinoids suggest the possibility of receptor subtypes. Two cannabinoid receptors have been identified to date; one is located predominantly in the central nervous system (CB1), whereas the other is expressed in peripheral tissues (CB2). The goal of this project is to characterize the pharmacology of subtypes of the cannabinoid receptor in order to elucidate the role of the receptors in vivo. We will use stably transfected cell lines which express cDNA clones of the individual receptor subtypes to provide a pure, homogeneous population of receptors for screening drug candidates. In specific aim 1, the binding and signal transduction properties of transfected cells expressing CB1 or CB2 cannabinoid receptor subtypes will be characterized. These studies will identify receptor subtype-selective ligands which discriminate between the receptor subtypes in binding and functional assays. The molecular mechanisms by which agonists bind to the cannabinoid receptors are not known. Specific aim 2 will examine which domains of the CB1 or CB2 receptors recognize the ligands and which activate second messenger systems. These studies will be conducted by site-directed mutagenesis and the construction of CB1/CB2 chimeric receptors in order to map the regions of the receptor cDNAs critical to receptor recognition and second messenger function. In specific aim 3, additional putative cannabinoid receptors will be isolated by polymerase chain reaction and library screening techniques. These studies will use the cumulative information obtained from the currently isolated receptor clones to isolate other subtypes which may provide data for the isolation of yet additional cannabinoid receptor subtypes. In summary, these experiments describe a molecular biological approach to studying cannabinoid receptor function. These studies will facilitate the design of new therapeutic strategies involving the cannabinoid system. Furthermore, insight into the molecular mechanisms of activation of cannabinoid receptors may lead to a better understanding of cannabinoid abuse in humans.